The present invention relates generally to Werner""s Syndrome and more specifically to methods and compositions suitable for use in diagnosis and treatment of Werner""s Syndrome.
Werner Syndrome (WS) is an autosomal recessive disorder with a complex phenotype. The disorder manifests itself in premature occurrence of age-related diseases and premature appearance of some of the physical features of normal aging. The onset of symptoms usually occurs after adolescence. The disorder progresses throughout life and typically patients have a shortened life expectancy with a modal age of death at 47. The prevalence of Werner Syndrome is estimated for heterozygotes to be 1-5 per 1,000 individuals, and for homozygotes to be 1-22 per 1,000,000 individuals.
Clinical symptoms of Werner Syndrome include both a prevalence of age-related diseases and physical features of aging. Such diseases include arteriosclerosis and heart disease, both benign and malignant neoplasms (usually sarcomas), diabetes mellitus, osteoporosis, and ocular cataracts. The physical appearance of WS patients is often manifest as a short stature, premature graying or loss sarcomas), diabetes mellitus, osteoporosis, and ocular cataracts. The physical appearance of WS patients is often manifest as a short stature, premature graying or loss of hair, hypogonadism, altered skin pigmentation, hyperkeratosis, tight skin, bird-like facies, cutaneous atrophy, cutaneous leg ulcers, and telangiectasia. Most of these diseases and features are present in from 40-90% of WS patients. Diagnosis of WS relies mainly upon the appearance of a certain number of these diseases and features. One biochemical test, excessive excretion of hyaluronic acid in urine, may also be used to assist diagnosis.
In addition to the noted signs and symptoms of aging, Werner Syndrome mimics normal aging as evidenced by the replicative potential of fibroblasts isolated from WS subjects. Replication potential of fibroblasts is reduced in these patients compared to fibroblasts isolated from age-matched controls, and is comparable to the replicative potential of fibroblasts taken from elderly subjects. Moreover, an increased mutation rate has been described in WS patients. Such abnormality is manifest as chromosomal instability, such as inversions, reciprocal translocations, deletions, and pseudodiploidy, and as increased mutation rate at the hypoxanthine phosphoribosyl transferase (HPRT) gene.
Werner Syndrome has been recognized as an autosomal recessive disorder. Goto et al. (Goto et al., Nature 355:735-738, 1992) mapped the WS gene onto the short arm of chromosome 8, using 21 affected Japanese families. The gene is located between marker D8S87 and ankyrin (ANK1). More recently, more refined mapping has pinpointed the WS gene to a region between marker D8S131 and D8S87, an 8.3 cM interval. Identification of the gene and gene product should add considerably to understanding the basis of Werner Syndrome and enable biochemical and genetic approaches to diagnosis and treatment.
The present invention provides a novel, previously unidentified gene for Werner Syndrome and compositions for diagnosis and treatment of WS, and further provides other related advantages.
Briefly stated, the present invention provides isolated nucleic acid molecules encoding the WRN gene, as well as portions thereof, representative of which are provided in the Figures. The protein which is encoded by the WRN gene is referred to hereinafter as the xe2x80x9cWRN proteinxe2x80x9d. Within other embodiments, nucleic acid molecules are provided which encode a mutant WRN gene product that increases the probability of Werner""s Syndrome (in a statistically significant manner). Representative illustrations of such mutants are provided in Example 3.
Within other aspects of the present invention, isolated nucleic acid molecules are provided, selected from the group consisting of (a) an isolated nucleic acid molecule as set forth in the Figures, or complementary sequence thereof, (b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecule of (a) under conditions of high stringency, and (c) an isolated nucleic acid that encodes a WRN gene product (WRN protein). As utilized herein, it should be understood that a nucleic acid molecule hybridizes xe2x80x9cspecificallyxe2x80x9d to an WRN gene (or related sequence) if it hybridizes detectably to such a sequence, but does not significantly or detectably hybridize to the Bloom""s Syndrome gene (Ellis et al., Cell 83:655-666, 1995).
Within other aspects, expression vectors are provided comprising a promoter operably linked to one of the nucleic acid molecule described above. Representative examples of suitable promoters include tissue-specific promoters, as well as promoters such as the CMV I-E promoter, SV40 early promoter and MuLV LTR. Within related aspects, viral vectors are provided that are capable of directing the expression of a nucleic acid molecule as described above. Representative examples of such viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors and retroviral vectors. Also provided are host cells (e.g., human, dog, monkey, rat or mouse cells) which carry the above-described vectors.
Within other aspects of the present invention, isolated proteins or polypeptides are provided comprising a WRN gene product, as well as peptides of greater than 12, 13 or 20 amino acids. Within another embodiment, the protein is a mutant WRN gene product that increases the probability of Werner""s Syndrome.
Within yet another aspect of the present invention, methods of treating or preventing Werner""s Syndrome are provided (as well as for related diseases which are discussed in more detail below), comprising the step of administering to a patient a vector containing or expressing a nucleic acid molecule as described above, thereby reducing the likelihood or delaying the onset of Werner""s Syndrome (or the related disease) in the patient. Within a related aspect, methods of treating or preventing Werner""s Syndrome (and related diseases) are provided, comprising the step of administering to a patient a protein as described above, thereby reducing the likelihood or delaying the onset of Werner""s Syndrome (or a related disease) in the patient. Within certain embodiments, the above methods may be accomplished by in vivo administration.
Also provided by the present invention are pharmaceutical compositions comprising a nucleic acid molecule, vector, host cell, protein, or antibody as described above, along with a pharmaceutically acceptable carrier or diluent.
Within other aspects of the present invention, antibodies are provided which specifically bind to an WRN protein or to unique peptides derived therefrom. As utilized herein, it should be understood that an antibody is specific for an WRN protein (or peptide) if it binds detectably, and with a Kd of 10xe2x88x927M or less (e.g., 10xe2x88x928M, 10xe2x88x929M, etc.), but does not bind detectably (or with an affinity of greater than 10xe2x88x927M, (e.g., 10xe2x88x926M, 10xe2x88x925M, etc.) to an unrelated helicase (e.g., the Bloom""s Syndrome gene, supra). Also provided are hybridomas which are capable of producing such antibodies.
Within other aspects of the present invention, nucleic acid probes are provided which are capable of specifically hybridizing (as defined below) to an WRN gene under conditions of high stringency. Within one related aspect, such probes comprise at least a portion of the nucleotide sequence shown in the Figures, or its complementary sequence, the probe being capable of specifically hybridizing to a mutant WRN gene under conditions of high stringency. Representative probes of the present invention are generally at least 12 nucleotide bases in length, although they may be 14, 16, 18 bases or longer. Also provided are primer pairs capable of specifically amplifying all or a portion of any of the nucleic acid molecules disclosed herein.
Within other aspects of the invention, methods are provided for diagnosing a patient having an increased likelihood of contracting Werner""s Syndrome (or a related disease), comprising the steps of (a) obtaining from a patient a biological sample containing nucleic acid, (b) incubating the nucleic acid with a probe which is capable of specifically hybridizing to a mutant WRN gene under conditions and for time sufficient to allow hybridization to occur, and (c) detecting the presence of hybridized probe, and thereby determining that said patient has an increased likelihood of contracting Werner""s Syndrome (or a related disease). Within another aspect, methods are provided comprising the steps of (a) obtaining from a patient a biological sample containing nucleic acid, (b) amplifying a selected nucleic acid sequence associated with a mutant WRN gene, and (c) detecting the presence of an amplified nucleic acid sequence, and thereby determining that the patient has an increased likelihood of contracting Werner""s Syndrome (or a related disease). Suitable biological samples include nucleated cells obtained from the peripheral blood, from buccal swabs, or brain tissue.
Within another aspect, peptide vaccines are provided which comprise a portion of a mutant WRN gene product containing a mutation, in combination with a pharmaceutically acceptable carrier or diluent.
Within yet another aspect, transgenic animals are provided whose germ cells and somatic cells contain a WRN gene (or lack thereof, i.e., a xe2x80x9cknockoutxe2x80x9d) which is operably linked to a promoter effective for the expression of the gene, the gene being introduced into the animal, or an ancestor of the animal, at an embryonic stage. Within one embodiment, the animal is a mouse, rat or dog. Within other embodiments, the WRN gene is expressed from a vector as described above. Within yet another embodiment, the WRN gene encodes a mutant WRN gene product.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.